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The topic of the research paper is Protein Aggregation
Irwin, J. (2012). Different Fates of Alzheimer’s Disease Amyloid-β Fibrils Remodeled by Biocompatible Small Molecules. Bio Macromolecules.http://dx.doi.org/10.1021/bm3016994
Abstract
This research paper investigates the effects of presence of three small biocompatible molecules on the formation of amyloid-beta (Aβ) aggregates. Methylene blue (MB), Brilliant Blue G (BBG), and Erythrosine B (ER) all lead to various Aβ aggregates.
Introduction
Formation of amyloid fibrils is important due to its crucial role in a number of neurodegenerative diseases.
Aβ peptide aggregates are characterized by thermodynamic stability. As a result, there is a lot of research done in order to figure out ways for the reversing or stopping the aggregation processes.
Small biological molecules have potential to affect the formation of amyloid fibrils in physiological conditions.
The three molecules that were picked for the experiments were chosen because they are non-toxic to humans and can be used for therapeutic application. In addition, all three molecules have the structures that are different enough to investigate the various effects of the molecules on aggregation of amyloid fibrils.
Experimental Procedures
All the materials for the experiments were purchased from various places.
The aggregation of the Aβ fibrils was performed under controlled conditions using established procedure.
The Thioflavin T Assay was used to verify the presence of Aβ aggregates during the experiments.
Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) were used to measure the widths and lengths of the aggregates.
Circular Dichroism (CD) measurements were used to analyze the secondary structure of the Aβ aggregates for all the samples; both treated and untreated with small biological molecules.
Previously reported Antibody Dot-Blot Assay was used to compare the interactions of the final Aβ aggregates with specified antibodies.
Results and Discussion
The data for untreated Aβ aggregation sample was collected to be used as the baseline for comparison. The Thioflavin T fluorescence verified that Aβ fibrils formed in the untreated sample after incubation. Then the TEM, AFM, and CD data were collected for the untreated sample.
Then the same analytical data was collected for the sample treated with BBG molecule. The analysis of the data showed that Aβ fibrils did form; however, the size of the fibrils was smaller than that of the untreated sample. Both AFM and TEM data verified the shorter size of the fibrils of Aβ aggregates.
Closer comparison of the widths and lengths of the fibrils that was collected using microscopy, led to the hypothesis that the Aβ aggregates that were treated with BBG molecules were the fragments of fibrils and not protofibrils.
In order to verify the hypothesis, CD data was collected and compared to the data of the untreated sample. The collected CD spectra verified the β-sheet structure of the sample treated with BBG. In addition, it reinforced the hypothesis that the treated sample has shorter Aβ fibril than the untreated one but the structure of the fibrils is the same.
The antibody dot-blot assay showed that the sample treated with BBG molecule did not affect the interactions of the final Aβ aggregates with the antibodies. As a result, it was concluded that the sample that is treated with BBG still results in the formation of Aβ fibril just shorter in size than the sample untreated with the molecule.
Then the sample that was treated with MB was analyzed. The microscopy data showed no evidence of the fibril formation and therefore no measurements of widths or lengths were taken. As a result, the analysis of the final aggregate was done using CD spectra collected. The spectra showed absence of β-sheet structure. The sample treated with MB molecule resulted in the formation of disordered amorphous aggregate.
Due to the lack of reference data for distorted and random protein aggregates, the CD spectra was only partially analyzed based on the estimated values of the DichroWeb server.
The analysis of the antibody dot-blot assay showed that the Aβ aggregates that was treated with MB lost most of its reactivity with the antibodies in comparison to the untreated sample. This led to a conclusion that MB prevents the formation of Aβ fibrils and leads to distorted amorphous aggregate instead.
Finally, the sample what was treated with ER was analyzed along with all the other aggregates. The analysis of the microscopy data showed that there were Aβ aggregates formed that have structure similar to fibrils. Quantitative comparison of the structures suggested that the final aggregates might be protofibrils.
Closer comparison of the widths of the Aβ aggregates treated with ER and measured using AFM showed that the final structure is most likely protofibrils unlike the BBG treated sample.
Analysis of CD spectra suggested that the sample treated with ER yielded to aggregates that have typical β-sheet structure.
The dot-blot assays results verified that the formed aggregates were indeed the protofibrils for the sample of amyloid peptide that was treated with ER molecules. It was also concluded that the different charges on the MB and ER molecules are responsible for the different aggregate formation when added to amyloid-beta fibrils.
Conclusion
In conclusion, the effects of three different molecules on the formation of Aβ aggregates were evaluated in the paper. All the molecules led to formation of different aggregates after being added to the samples of Aβ fibrils. BBG molecules led to formation of shorter Aβ fibrils, which retained the same structure as the untreated sample. MB molecules resulted in the formation of amorphous aggregate, which completely distorted the β-sheet structure of the peptide. However, ER molecules led to formation of protofibrils after being added to the peptide. As a result, all three molecules have potential to be used to interfere in the process of aggregation.
The topic of the research paper is Protein Aggregation
Irwin, J. (2012). Different Fates of Alzheimer’s Disease Amyloid-β Fibrils Remodeled by Biocompatible Small Molecules. Bio Macromolecules. http://dx.doi.org/10.1021/bm3016994
Abstract
This research paper investigates the effects of presence of three small biocompatible molecules on the formation of amyloid-beta (Aβ) aggregates. Methylene blue (MB), Brilliant Blue G (BBG), and Erythrosine B (ER) all lead to various Aβ aggregates.
Introduction
Formation of amyloid fibrils is important due to its crucial role in a number of neurodegenerative diseases.
Aβ peptide aggregates are characterized by thermodynamic stability. As a result, there is a lot of research done in order to figure out ways for the reversing or stopping the aggregation processes.
Small biological molecules have potential to affect the formation of amyloid fibrils in physiological conditions.
The three molecules that were picked for the experiments were chosen because they are non-toxic to humans and can be used for therapeutic application. In addition, all three molecules have the structures that are different enough to investigate the various effects of the molecules on aggregation of amyloid fibrils.
Experimental Procedures
All the materials for the experiments were purchased from various places.
The aggregation of the Aβ fibrils was performed under controlled conditions using established procedure.
The Thioflavin T Assay was used to verify the presence of Aβ aggregates during the experiments.
Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) were used to measure the widths and lengths of the aggregates.
Circular Dichroism (CD) measurements were used to analyze the secondary structure of the Aβ aggregates for all the samples; both treated and untreated with small biological molecules.
Previously reported Antibody Dot-Blot Assay was used to compare the interactions of the final Aβ aggregates with specified antibodies.
Results and Discussion
The data for untreated Aβ aggregation sample was collected to be used as the baseline for comparison. The Thioflavin T fluorescence verified that Aβ fibrils formed in the untreated sample after incubation. Then the TEM, AFM, and CD data were collected for the untreated sample.
Then the same analytical data was collected for the sample treated with BBG molecule. The analysis of the data showed that Aβ fibrils did form; however, the size of the fibrils was smaller than that of the untreated sample. Both AFM and TEM data verified the shorter size of the fibrils of Aβ aggregates.
Closer comparison of the widths and lengths of the fibrils that was collected using microscopy, led to the hypothesis that the Aβ aggregates that were treated with BBG molecules were the fragments of fibrils and not protofibrils.
In order to verify the hypothesis, CD data was collected and compared to the data of the untreated sample. The collected CD spectra verified the β-sheet structure of the sample treated with BBG. In addition, it reinforced the hypothesis that the treated sample has shorter Aβ fibril than the untreated one but the structure of the fibrils is the same.
The antibody dot-blot assay showed that the sample treated with BBG molecule did not affect the interactions of the final Aβ aggregates with the antibodies. As a result, it was concluded that the sample that is treated with BBG still results in the formation of Aβ fibril just shorter in size than the sample untreated with the molecule.
Then the sample that was treated with MB was analyzed. The microscopy data showed no evidence of the fibril formation and therefore no measurements of widths or lengths were taken. As a result, the analysis of the final aggregate was done using CD spectra collected. The spectra showed absence of β-sheet structure. The sample treated with MB molecule resulted in the formation of disordered amorphous aggregate.
Due to the lack of reference data for distorted and random protein aggregates, the CD spectra was only partially analyzed based on the estimated values of the DichroWeb server.
The analysis of the antibody dot-blot assay showed that the Aβ aggregates that was treated with MB lost most of its reactivity with the antibodies in comparison to the untreated sample. This led to a conclusion that MB prevents the formation of Aβ fibrils and leads to distorted amorphous aggregate instead.
Finally, the sample what was treated with ER was analyzed along with all the other aggregates. The analysis of the microscopy data showed that there were Aβ aggregates formed that have structure similar to fibrils. Quantitative comparison of the structures suggested that the final aggregates might be protofibrils.
Closer comparison of the widths of the Aβ aggregates treated with ER and measured using AFM showed that the final structure is most likely protofibrils unlike the BBG treated sample.
Analysis of CD spectra suggested that the sample treated with ER yielded to aggregates that have typical β-sheet structure.
The dot-blot assays results verified that the formed aggregates were indeed the protofibrils for the sample of amyloid peptide that was treated with ER molecules. It was also concluded that the different charges on the MB and ER molecules are responsible for the different aggregate formation when added to amyloid-beta fibrils.
Conclusion
In conclusion, the effects of three different molecules on the formation of Aβ aggregates were evaluated in the paper. All the molecules led to formation of different aggregates after being added to the samples of Aβ fibrils. BBG molecules led to formation of shorter Aβ fibrils, which retained the same structure as the untreated sample. MB molecules resulted in the formation of amorphous aggregate, which completely distorted the β-sheet structure of the peptide. However, ER molecules led to formation of protofibrils after being added to the peptide. As a result, all three molecules have potential to be used to interfere in the process of aggregation.